Reagents List

Product Name	Cat.NO.
Hieff Trans™ Booster DNA/RNA Transfection Reagent	40801ES

Sf9 -DNA Transfection Protocol

Cell Culture and Passaging (T25 Flask)

• Cell Counting: Remove cells from the incubator, gently mix the cell suspension by pipetting, and use a cell counter to determine cell viability and density.
• Cell Passage (Direct Dilution Method): Based on the counting results, passage the cells by diluting to a concentration of approximately 5 × 10⁶ cells/mL.
• Cell Passage (Half-medium Replacement Method): If the culture supernatant is noticeably yellow, after removing the cells, keep them undisturbed to allow cells to settle. Tilt the culture vessel slightly and carefully aspirate the old supernatant using a pipette. Then add fresh medium, mix well, and proceed with counting and passaging.
• Cell Passage (Centrifugation Method): If the cells are in poor condition, passage via low-speed centrifugation. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 200 ×g for 3–5 minutes. Aspirate the supernatant, resuspend the cell pellet in fresh culture medium, and then count and passage the cells.

Cell Transfection (6-Well Plate)

I. Preparation

1. Cell Preparation: 24 hours before transfection, take the cell suspension and perform cell counting. Passage the cells at a density of 5 × 10⁶ cells/mL, adding 2 mL of complete medium per well (referring to the total medium volume).

2. Reagent Preparation:

• Plasmid DNA: Use high-purity plasmid DNA (e.g., purified by kit), ideally at a concentration >1000 ng/μL.  
• Transfection Reagent: Allow Hieff Trans™Booster Transfection Reagent to warm to room temperature. Gently mix before use.  
• Serum-Free Medium: Prepare serum- and antibiotic-free basal medium (e.g., Opti-MEM) for diluting DNA and transfection reagent.

II. Complex Formation

1. Dilute DNA: In a sterile microcentrifuge tube, add 125 μL serum-free medium followed by 2.5 μg plasmid DNA. Mix gently. Then add 5 μL Enhancer, and mix again to obtain diluted DNA.  

2. Dilute Transfection Reagent: In another sterile tube, add 125 μL serum-free medium and 5 μL Hieff Trans™Booster Transfection Reagent. Mix gently.  

3. Combine and Incubate: Add the diluted DNA solution to the diluted transfection reagent tube. Mix gently by pipetting. Incubate at room temperature for 10–15 minutes to form DNA-transfection reagent complexes.

III. Transfection

1. Add Complexes: Add the incubated DNA-transfection reagent complexes (~250 μL total) dropwise and evenly to the cells. Gently rock the plate to distribute.  

2. Incubation: Return cells to a 37°C, 5% CO₂ incubator for culture.

IV. Post-Transfection Handling

1. Medium Replacement: 4–6 hours post-transfection, check cell morphology. If significant toxicity is observed, aspirate the medium containing complexes and replace with 2 mL fresh pre-warmed complete medium. If cells appear healthy, medium change is optional.  

2. Analysis: After 24–72 hours, assess transfection efficiency or functional outcomes based on experimental design (e.g., fluorescence observation, mRNA or protein detection).

Tips:

1. If significant cytotoxicity occurs, replace medium after 6 h or reduce Enhancer volume by half to mitigate toxicity.  

2. If transfection efficiency is low, increasing the amount of transfection reagent may improve results.  

3. For optimal performance, dilute DNA and transfection reagent in Opti-MEM rather than DMEM.  

4. The transfection system is compatible with serum and antibiotics; however, DNA and reagent dilutions must be performed in serum- and antibiotic-free medium.

Experimental Results Analysis

Transfection of plasmid DNA (6,738 bp) into Sf9 insect ovarian cells using Yeasen Booster DNA&RNA Transfection Reagent and L*2000 transfection reagent, followed by assessment of transfection efficiency using fluorescence microscopy.

Figure 1. Plasmid DNA transfection in sf9 using Hieff Trans™ Booster DNA&RNA Transfection Reagent versus L* 2000 transfection reagent. The result demonstrates superior cell transfection efficiency of Booster.

Different Cell Culture Vessel Transfection Volumes (for reference only):

Culture vessel	Medium Volume		DNA Transfection			siRNA Transfection (Final Concentration 50 nM)
	Volume of Medium	Volume of Opti-MEM Complex	DNA (μg)	Booster Transfection Reagent (μL)	Transfection Enhancer (μL)	Volume of siRNA (Initial Concentration 20 μM)	Booster Transfection Reagent (μL)
96-well	100 μL	2×5 μL	0.1	0.2	0.2	0.25 μL	0.3
48-well	250 μL	2×12.5 μL	0.25	0.5	0.5	0.625 μL	0.75
24-well	500 μL	2×25 μL	0.5	1	1	1.25 μL	1.5
12-well	1 mL	2×50 μL	1	2	2	2.5 μL	3
6-well	2 mL	2×125 μL	2.5	5	5	5 μL	7.5
60 mm	5 mL	2×250 μL	5-10	10-20	10-20	12.5 μL	20
10 cm	10 mL	2×500 μL	15-25	30-50	30-50	25 μL	40
T25	6 mL	2×250 μL	6-12	12-24	12-24	15 μL	24
T75	15 mL	2×750 μL	20-40	40-80	40-80	37.5 μL	60

[Note]: The volumes provided in this table are for reference only and are suitable for both suspension and adherent cells. For suspension cells, the test can be performed at a cell density of 0.5–1×10⁶ cells/mL. The actual amounts of DNA and Booster DNA/RNA transfection reagent should be optimized depending on cell type and other experimental conditions. It is recommended to maintain a ratio between 1:0.5 and 1:5 (DNA : Booster transfection reagent). The amount and experimental conditions for mRNA are the same as those for DNA. If the cells are particularly fragile and excessive cell death is observed, better results may be achieved by reducing the amount of enhancer by half.

 

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